Comparison of Rosco Neo-Sensitabs with Oxoid paper disks in EUCAST disk diffusion antimicrobial susceptibility testing on Mueller-Hinton agar.

This study compared Neo-Sensitabs with Oxoid paper disks using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) disk diffusion antimicrobial susceptibility test on Mueller-Hinton agar. The EUCAST-recommended quality control strains (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212) (Part I) and clinical isolates (Part II) were investigated. In Part I of the study, 27 combinations of antimicrobial agents were tested on four quality control strains repeatedly up to 60 times and zone diameters of tablets and disks were compared. In Part II of the study, 351 clinical isolates were included to cover a broad range of species, as well as resistance mechanisms. In Part I, four major deviations (>1 mm outside quality control ranges) were observed with Neo-Sensitabs. In one case with P. aeruginosa ATCC 27853 (meropenem), there was a corresponding major deviation (2 mm) with the Oxoid disk. The three remaining major deviations with Neo-Sensitabs were observed with meropenem (2 mm) in E. coli ATCC 25922 and with ciprofloxacin (2 mm) and gentamicin (3 mm) in P. aeruginosa ATCC 27853. For Oxoid disks, there were only minor deviations (=1 mm outside quality control ranges) in these three cases. In Part II, there were six discrepancies, susceptible versus resistant, in 3,533 comparisons between the two methods with the clinical isolates. The Rosco Neo-Sensitabs appear to be a possible alternative to Oxoid paper disks for EUCAST disk diffusion antimicrobial susceptibility testing on Mueller-Hinton agar.

Intracellular persistence of Escherichia coli in urinary bladders from mecillinam-treated mice.

OBJECTIVES: It has been suggested recently that intracellular bacteria surviving antibiotic treatment might serve as a reservoir for recurrent infection. The purpose of this study was to directly examine the location of Escherichia coli bacteria in the mouse bladder after treatment with mecillinam. METHODS: The bladders were studied by use of colony counts, in situ hybridization and electron microscopy. RESULTS: The bacterial counts in the bladder remained approximately 10(3-4) cfu/bladder even after mecillinam treatment had finished, and re-growth in the urine was observed. In the bladder epithelium from treated mice, bacteria cells were occasionally seen, presumably representing intracellularly located bacteria. CONCLUSIONS: This is the first in vivo study indicating that during mecillinam treatment E. coli cells can penetrate the mouse bladder epithelium and persist.

The effect of broth media on pneumococcal growth and the latex serotyping result.

The aim was to test Todd-Hewitt broths (TH-broths) for the ability to propagate pneumococci and thereafter to evaluate the serotyping result obtained by the Pneumotest-Latex kit (SSI). TH-broths from four different producers (Oxoid, Sigma, Difco, and SSI) were tested and compared separately and with Serum broth (SSI). Twenty-three pneumococcal strains (different serotypes) were inoculated into the broths with start inoculums of 10(1), 10(3), and 10(6) CFU/ml. After incubation, overnight viable bacterial counts and visible growth were recorded. All pneumococci were serotyped with the Pneumotest-Latex kit. After incubation, the bacterial counts in all TH-broths were within the range of log 4.65-log 7.76 CFU/ml, while Serum broth showed an average growth ranging from log 8.05-log 8.90 CFU/ml. Comparing the growth of the four TH-broths showed no significant differences. In general, Serum broth had a more pronounced visual growth than each of the four TH-broths. Serotyping with Serum broth showed in general positive and correct latex typing results for all serotypes and initial inoculum, while the outcome of the TH-broths showed some false negative results depending on inoculum and serotype. Overall the Serum broth was found to be superior to the four TH-broths tested both with regard to CFU/ml and when used with the Pneumotest-Latex kit. However, if the Pneumotest-latex kit is only used on broths with visible growth as stated in the instruction manual, then the differences between the performances of the broths from the different producers was not significant and all broths could be used for Pneumotest-Latex typing.

Simple, rapid latex agglutination test for serotyping of pneumococci (Pneumotest-Latex).

The "gold standard" for epidemiological typing of Streptococcus pneumoniae (pneumococcus) is the capsular reaction test (Neufeld test) with antisera against the 90 pneumococcal polysaccharide capsules, i.e., serotyping. The method is labor intensive and requires a certain level of experience to be performed satisfactory, and thus it has been restricted for use in specialized reference or research laboratories. Surveillance of the serotype distribution of pneumococci that cause infections is important to secure an optimal composition of pneumococcal vaccines and to monitor antibiotic resistance in pneumococci. At Statens Serum Institut, a simple latex agglutination test for serotyping of pneumococci has been developed. The Pneumotest-Latex kit consists of 14 different pooled pneumococcus antisera (pools A to I and pools P to T) applied to latex particles. In a blind test of 352 isolates (with all 90 serotypes represented), 336 (95.5%) were typed or grouped correctly by the Pneumotest-Latex; in addition, 2 (7%) of 30 strains regarded as nontypeable or rough strains were serotyped, and the serotypes of these two isolates were confirmed by the capsular reaction test with type-specific antisera. The Pneumotest-Latex seems to be a sensitive method for serotyping or grouping of the majority of pneumococcal strains. By use of this ready-to-perform latex agglutination kit (Pneumotest-Latex), serotyping of pneumococci can gain more ground as a tool in prevention of pneumococcal diseases.

Urinary concentrations and urine ex-vivo effect of mecillinam and sulphamethizole.

Healthy adult volunteers received 1 g of sulphamethizole orally (n = 10) and later 400 mg of pivmecillinam (274 mg of mecillinam) (n = 9). All urine was collected in defined periods over 24 h, and the drug concentrations in urine were determined. For sulphamethizole, the maximum urine concentration for seven subjects was reached in 0-3 h, and for the remaining three in 3-6 h. For mecillinam, eight of the nine subjects attained a maximum urine concentration in 0-3 h, after which the concentration declined rapidly for six subjects in 3-6 h. Strains of Escherichia coli with different MICs for sulphamethizole and mecillinam were exposed to collected urine for 2.5 h and 5 h. The results indicated that a sensitive E. coli population should be suppressed by sulphamethizole in urine for two-thirds of the time (with 1 g twice-daily) and by mecillinam in urine throughout the 24-h period (with 400 mg three times a day). There was a slight but significant correlation between the ex-vivo effect (Delta log10 CFU/mL) and the log10 concentration/MIC ratio after exposure to sulphamethizole for 5 h (r2 = 0.27, p < 0.0001), and a significant correlation between the variables with mecillinam (r2 = 0.66, p < 0.0001).

Effects of sulfamethizole and amdinocillin against Escherichia coli strains (with various susceptibilities) in an ascending urinary tract infection mouse model.

Resistance to antibiotics used for the treatment of urinary tract infections (UTIs) is increasing worldwide. The impact of in vitro resistance on clinical outcome in UTIs requires further study, since most studies of both humans and animals have evaluated only the efficacy of antibiotics toward bacteria susceptible in vitro. We were interested in evaluating the relationship between the in vitro antibacterial effect and the in vivo efficacy after antibiotic treatment. We simulated a natural ascending UTI by use of the ascending UTI mouse model and used Escherichia coli strains with various susceptibilities to amdinocillin (mecillinam) and sulfamethizole. Mice were treated for 3 days with antibiotic doses approximating human urinary tract concentrations after a standard oral dose. For a susceptible strain (MIC, 0.5 micro g/ml) and a resistant strain (MIC, 128 micro g/ml), respectively, there were significant reductions in bacterial counts in the urine, bladder, and kidneys after treatment with amdinocillin, whereas for a strain for which the MIC was 16 micro g/ml, there was a significant reduction in bacterial counts in the kidneys only (P < 0.05). Treatment with sulfamethizole resulted in a significant reduction in bacterial counts in all samples from a susceptible strain (MIC, 128 micro g/ml) and a resistant strain (MIC, 512 micro g/ml). Infection with a sulII gene-positive strain (MIC, >2,048 micro g/ml) could not be treated with sulfamethizole, as no effect could be demonstrated in the urine, bladder, or kidneys. For amdinocillin, there was no clear-cut relationship between the in vitro susceptibility and the in vivo outcome, while for sulfamethizole, we found a relationship between the MIC for the strain and the effect in the urinary tract.

Susceptibility of Danish Escherichia coli strains isolated from urinary tract infections and bacteraemia, and distribution of sul genes conferring sulphonamide resistance.

Antibiotic resistance of urinary tract pathogens has increased worldwide. Our aim was to provide information regarding resistance patterns of Escherichia coli in urinary tract infections (UTIs) and E. coli bacteraemia in Denmark. The overall resistance ranged from: ampicillin 20-47%, mecillinam 0-7%, trimethoprim 10-28%, sulfamethizole 22-47% and nitrofurantoin 0-3%. In strains with sulfamethizole MICs > 2048 mg/L, 97% carried sulI, sulII or both genes, with sulII being the most common. Among the sulI gene-positive strains, 96% were intI 1 gene positive.